Molecular dissection of the genes responsible for the formation of extracellular polysaccharide capsule in Cryptococcus neoformans, a major opportunistic fungal pathogen causing infection primarily in immunocompromised patients. The extracellular polysaccharide capsule of C. neoformans has been the major virulence factor of the organism. To understand the molecular aspects of capsule synthesis in c. neoformans, we attempted to clone the genes controlling the formation of polysaccharide capsule in the fungus. A wild type genomic DNA library of C. neoformans B- 3501 was used to complement various cap mutants. The plasmids containing appropriate DNA inserts from Cap transformants was rescued in E. coli. Subsequent complementation of various Cap mutants with the subclones of the insert DNA's indicated that we have cloned the gene, Cap59. To demonstrate that the Cap59 gene is essential for the virulence of C. neoformans, the gene was deleted from a wild -type strain and tested for its' virulence. The Cap59 deleted strain became avirulent in mice while the wild type strain produced fatal infection within 20 days in all 10 (100%) mice. The gene deletion by homologous integration presented in this study is the first such report in C. neoformans. A linear minichromosome of 270kb in size was found to have been created in a C. neoformans ura5 mutant as a result of transformation with a plasmid containing C. neoformans URA5 gene. The minichromosome was as stably maintained as native chromosomes during mitotic or meiotic cell cycle. Analysis of the minichromosome showed that is contains at least four copies of URA5 sequence and 23 copies of rDNA clusters in addition to unknown sequences. Analysis of the subclones of the unidentified DNA showed that there are several repeats of the conserved core of the ARS-like (autonomously replicating sequences), sequences [A/TTTTAT(A/G)TTT(A/T)] within a 4.5kb fragment. These sequences were suspected to be the sequences contributing to its apparent stability by the maintenance of high copy numbers of the host plasmid. The arrangement and sequence of the 55 rRNA in C. neoformans was determined. The gene is composed of 118 nucleotides and is located at 1kb upstream of the small subunit rRNA gene within the rRNA cluster. The 55 gene was found to be transcribed at the same direction as is the case of the 55 gene in the majority of the higher basidiomycetous fungi.